The availability of novel HDAC class- and isoform-specific PET radiotracers will have a significant positive impact on the pace or research in the field of epigenetics. SIRTs-specific PET imaging agents will enable non- invasive and repetitive in vivo imaging of SIRTs expression and activity in the brain and different organs and tissues (including cancer) and help to understand the mechanisms of SIRTs involvement in normal physiology and in the mechanisms of different diseases, enable non-invasive monitoring of pharmacodynamics and therapeutic efficacy of novel SIRTs-specific inhibitors (or activators) in experimental animals and in humans, and facilitate their translation into clinic. Therefore, the overall aim of this grant application is to conduct a series of comprehensive in vivo imaging studies in rodents and non-human primates to further assess the efficacy of 18F-PhFAHA for non-invasive imaging of SIRTs in the brain and other organs and tissues. To further improve substrate selectivity and binding to SIRTs, we will synthesize and evaluate 42 potential SIRTs-specific substrates different 18F-labeled acyl leaving groups and two different side chains in the cap region. The substrate efficiency of compounds in the focused library will be in a panel of recombinant HDACs 1-11 and SIRTs 1-7. Three best substrates will be radiolabeled with F-18 and evaluated further in vitro and in vivo. Then, In vitro radiotracer accumulation studies will be performed with 18F-PhAHA and three novel 18F-labeled radiotracers in tumor cell lines with high, moderate, and low levels of SIRT1 expression; we will determine the radiolabeled metabolites in the primary cultures of human hepatocytes. By conducting these in vitro studies we will select three best novel radiotracers for subsequent microPET/CT imaging and autoradiography studies in mice to assess their efficacy and specificity for imaging SIRTs expression-activity. We will perform in situ immunohistochemical comparative studies to validate PET/CT imaging results. Also, PET/CT imaging studies will be performed in tumor-bearing mice at: a) baseline; b) pre-treatment with resveratrol (SIRTs activator); c) pre-treatment with sirtinol (SIRTs inhibitor) to assess the feasibility of pharmacodynamic imaging. The best radiotracer will be selected for subsequent studies in non-human primates to assess: a) the time-course of biodistribution, uptake, retention, and efflux from different organs and tissues; b) whole body and individual organ radiation dosimetry; and d) the time-course of 18F-labeled metabolites in blood. Also, we will assess the feasibility of PET/CT/MRI with the selected novel radiotracer (or 18F-PhAHA) for monitoring pharmacodynamic (PD) effects of SIRTs activators and inhibitors (resveratrol and sirtinol) in the same animals. Through the proposed studies we will select SIRTs-specific 18F-labeled radiotracer with optimal pharmacokinetic and radiation dosimetry characteristics and sensitivity for PET/CT/MRI imaging of SIRTs expression-activity in vivo.